The carboxy-terminal domain of the alpha subunit of RNAP (a-CTD) plays an important role in transcriptional activation in Escherichia coli. To better understand transcriptional activation, we investigated the interaction of the a-CTD with MarA, a transcriptional activator of resistance to multiple antibiotics, superoxides and organic solvents. We identified the interacting surfaces of MarA with the a-CTD by NMR-based chemical shift mapping. Spectral changes were monitored for a MarA-DNA complex upon titration with a-CTD, and for a-CTD upon titration with MarA-DNA. The mapping results were confirmed by mutational studies and retention chromatography. A model of the ternary complex shows that a-CTD uses a ?265-like determinant? to contact MarA at a surface distant from the DNA. This is unlike the interaction of a-CTD with the CRP or Fis activators where the ?265 determinant? contacts DNA while another surface of the same a-CTD molecule contacts the activator. These results reveal a new versatility for a-CTD in transcriptional activation. It has been observed by others that mar operon mRNA accumulates in cells treated with cerain antibiotics which they have taken to mean that these antibiotics derepress the operon. We have used several methods to show that this is likely to be an artifact since in vivo there is no sign of increased expression of the operon following antibiotic treatment.